Nociceptin Opioid Peptide Receptor Research Articles

An examination of the effects of nucleus accumbens core nociceptin on appetitive and consummatory motivation for food

The nucleus accumbens (NAc) is a critical region for regulating the appetitive and consummatory aspects of motivated behavior. Previous work has shown differential effects of NAc µ-, δ-, and κ- receptor stimulation on food intake and for shifting motivation within an effort-based choice (EBC) task. However, the motivational role of the nociceptin opioid peptide (NOP) receptor, a fourth member of the opioid receptor family, is less well understood. These experiments therefore characterized the effect of NAc injections of nociceptin, the endogenous ligand for the NOP receptor, on consummatory and appetitive motivation. Three groups of male Sprague-Dawley rats received nociceptin injections into the NAc core prior to testing in a progressive ratio lever pressing task, an EBC task, or a palatable feeding assay. In the feeding experiment, 10 nmol of nociceptin increased consumption in the first 30 min, but this increase was not sustained through the end of the 2-hr session. Additionally, nociceptin injections did not alter breakpoint in the progressive ratio task. However, in the EBC task, nociceptin significantly decreased breakpoint for sugar pellets without affecting consumption of rat chow. These data suggest that NAc NOP receptor stimulation transiently increases consummatory motivation toward palatable diets and inhibits appetitive motivation when alternate food options are freely available. This pattern of effects contrasts with those obtained following NAc stimulation of other opioid receptors, suggesting that the four opioid receptor classes each serve unique roles in modulating food-directed motivation within the NAc core.

Imaging Nociceptin Opioid Peptide Receptors in Alcohol Use Disorder With [11C]NOP-1A and Positron Emission Tomography: Findings From a Second Cohort

BackgroundNociceptin, which binds to the nociceptin opioid peptide receptor (NOP), regulates stress and reward in addiction. In a previous [11C]NOP-1A positron emission tomography (PET) study, we found no differences in NOP in non–treatment-seeking individuals with alcohol use disorder (AUD) relative to healthy control subjects Here, we evaluated NOP in treatment-seeking individuals with AUD to document its relationship with relapse to alcohol. Methods[11C]NOP-1A distribution volume (VT) was measured in recently abstinent individuals with AUD and healthy control subjects (n = 27/group) using an arterial input function–based kinetic analysis in brain regions that regulate reward and stress behaviors. Recent heavy drinking before PET was quantified using hair ethyl glucuronide (≥30 pg/mg was defined as heavy drinking). To document relapse, 22 subjects with AUD were followed with urine ethyl glucoronide tests (3/week) for 12 weeks after PET, where they were incentivized with money to abstain. ResultsThere were no differences in [11C]NOP-1A VT between individuals with AUD and healthy control subjects. Individuals with AUD who drank heavily before the study had significantly lower VT than those with no recent heavy drinking history. Significant negative correlations between VT and the number of drinking days and the number of drinks consumed per drinking day in the 30 days before enrollment were also present. Individuals with AUD who relapsed (and dropped out) had significantly lower VT than those who abstained for 12 weeks. ConclusionsLower NOP VT in heavy drinking AUD predicted relapse to alcohol during a 12-week follow-up period. The results of this PET study support the need to investigate medications that act at NOP to prevent relapse in individuals with AUD.

Current and Future Therapeutic Optionsin Pain Management: Multi-mechanistic Opioids Involving Both MOR and NOP Receptor Activation.

Opioids are widely used in chronic pain management, despite major concerns about their risk of adverse events, particularly abuse, misuse, and respiratory depression from overdose. Multi-mechanistic opioids, such as tapentadol and buprenorphine, have been widely studied as a valid alternative to traditional opioids for their safer profile. Special interest was focused on the role of the nociceptin opioid peptide (NOP) receptor in terms of analgesia and improved tolerability. Nociceptin opioid peptide receptor agonists were shown to reinforce the antinociceptive effect of mu opioid receptor (MOR) agonists and modulate some of their adverse effects. Therefore, multi-mechanistic opioids involving both MOR and NOP receptor activation became a major field of pharmaceutical and clinical investigations. Buprenorphine was re-discovered in a new perspective, as an atypical analgesic and as a substitution therapy for opioid use disorders; and buprenorphine derivatives have been tested in animal models of nociceptive and neuropathic pain. Similarly, cebranopadol, a full MOR/NOP receptor agonist, has been clinically evaluated for its potent analgesic efficacy and better tolerability profile, compared with traditional opioids. This review overviews pharmacological mechanisms of the NOP receptor system, including its role in pain management and in the development of opioid tolerance. Clinical data on buprenorphine suggest its role as a safer alternative to traditional opioids, particularly in patients with non-cancer pain; while data on cebranopadol still require phase III study results to approve its introduction on the market. Other bifunctional MOR/NOP receptor ligands, such as BU08028, BU10038, and AT-121, are currently under pharmacological investigations and could represent promising analgesic agents for the future.

PPL-138 (BU10038): A bifunctional NOP/mu partial agonist that reduces cocaine self-administration in rats

The search for new and effective treatments for cocaine use disorder (CUD) is a priority. We determined whether PPL-138 (BU10038), a compound with partial agonist activity at both nociceptin opioid peptide (NOP) and mu-opioid receptors, reduces cocaine consumption, reinstatement, and whether the compound itself produces reinforcing effects in rats. Using an intermittent access (IntA) cocaine self-administration procedure, we found that PPL-138 (0.1 and 0.3 mg/kg) effectively decreased the total number of cocaine infusions and burst-like cocaine intake in both male and female rats. Responses for food in an IntA model of food self-administration were not altered for either sex, although locomotor activity was increased in female but not male rats. Blockade of NOP receptors with the selective antagonist J-113397 (5 mg/kg) did not prevent the PPL-138-induced suppression of cocaine self-administration, whereas blockade of mu-opioid receptors by naltrexone (1 mg/kg) reversed such effect. Consistently, treatment with morphine (1, 3, and 10 mg/kg) dose-dependently reduced IntA cocaine self-administration measures. PPL-138 also reduced reinstatement of cocaine seeking at all doses examined. Although an initial treatment with PPL-138 (2.5, 10, and 40 μg/kg/infusion) appeared rewarding, the compound did not maintain self-administration behavior. Animals treated with PPL-138 showed initial suppression of cocaine self-administration, which was eliminated following repeated daily dosing. However, suppression of cocaine self-administration was retained when subsequent PPL-138 treatments were administered 48 h apart. These findings demonstrate that the approach of combining partial NOP/mu-opioid activation successfully reduces cocaine use, but properties of PPL-138 seem to depend on the timing of drug administration.

Lack of effect of the nociceptin opioid peptide agonist Ro 64-6198 on pain-depressed behavior and heroin choice in rats

Rationale and objectiveOne objective of the National Institutes of Health Helping to End Addiction Long-term (HEAL) initiative is to accelerate research on safer and more effective medications for both pain and opioid use disorder. Ligands that activate the nociceptin opioid peptide receptor (NOP) constitute one class of candidate drugs for both applications. The present preclinical study determined the effectiveness of the NOP agonist Ro 64-6198 to produce antinociception in a pain-depressed behavior procedure and attenuate opioid self-administration in a heroin-vs-food choice procedure. MethodsIn Experiment 1, Adult Sprague-Dawley rats were equipped with microelectrodes and trained to respond for electrical brain stimulation in an intracranial self-stimulation (ICSS) procedure. The potency, time course, and receptor mechanism of effects produced by R0 64-6198 alone (0.32–3.2 mg/kg) on ICSS were examined, followed by evaluation of 0.32–1.0 mg/kg Ro 64-6198 effectiveness to block lactic acid-induced depression of ICSS. In Experiment 2, rats self-administered heroin under a heroin-vs-food choice procedure during a regimen of repeated, daily intraperitoneal administration of vehicle or Ro 64-6198 (1–3.2 mg/kg/day). ResultsRo 64-6198 produced dose- and time-dependent ICSS depression that was blocked by the selective NOP antagonist SB612111 but not by naltrexone. Ro 64-6198 failed to block acid-induced depression of ICSS. Repeated Ro 64-6198 pretreatment also failed to attenuate heroin-vs-food choice up to doses that significantly decreased operant behavior. ConclusionsThese results do not support the utility of Ro 64-6198 as a stand-alone medication for either acute pain or opioid use disorder.

Cardiovascular and renal effects of novel nonpeptide nociceptin opioid peptide receptor agonists.

Partial agonists of the nociceptin opioid peptide (NOP) receptor have potential therapeutic use as antihypertensive and water diuretics (aquaretics). To date, peptide NOP receptor ligands have failed to progress in clinical trials due to poor pharmacokinetics and adverse effects. Nonpeptide, small-molecule NOP receptor ligands may be more suitable as therapeutic agents. This study investigated the cardiovascular and renal responses produced by the novel nonpeptide NOP agonists AT-403, AT-090, AT-127, and AT-039. Changes in mean arterial pressure (MAP), heart rate (HR), renal excretory function and occurrence of sedation and hyperphagia were determined before and after i.v. bolus injection or infusion of the NOP agonists in conscious Sprague-Dawley rats. Additional studies involving (i) measurement of renal sympathetic nerve activity (RSNA) and (ii) renal denervation were conducted to investigate the role of the renal nerves in the cardiorenal responses to AT-039. Bolus i.v. injection of AT-403, AT-090, AT-127 and AT-039 produced significant decreases in MAP and HR and a sodium-sparing diuresis. AT-403, AT-090, and AT-127, but not AT-039, induced sedation and hyperphagia at all doses tested. Infusion i.v. of AT-039 produced hypotension and aquaresis without adverse central nervous system effects or change in HR, responses that were also observed in renal denervated rats. Nonpeptide NOP agonists decrease blood pressure and produce aquaresis in conscious rodents. Due to lack of sedation and hyperphagia, AT-039 represents a novel NOP agonist that may be useful for treatment of hypertension and/or volume overload/hyponatraemic states.

Involvement of the nociceptin opioid peptide receptor in morphine-induced antinociception, tolerance and physical dependence in female mice.

Nociceptin opioid peptide (NOP) receptor modulates pain transmission and is considered a prospective target for pain management. Under acute pain conditions in rodents, however, no definitive conclusions about effects of systemically intervening NOP receptors on nociception, classical opioid-induced antinociception, tolerance and physical dependence have been drawn. Given that opioid analgesia has sex differences, and females experience greater pain and consume more opioids, clarifying these issues in females will help develop novel analgesics. To clarify the role of NOP receptors on the pharmacological profiles of µ-opioid receptor agonists, in this study, a selective agonist (SCH221510) and antagonist (SB612111) of the NOP receptor were subcutaneously administered in female mice in multiple animal models. In hot-plate test, neither SCH221510 (3 and 10mg/kg, sc) nor SB612111 (10mg/kg, sc) produced significant antinociception. SCH221510 (3mg/kg, sc) attenuated but SB612111 (10mg/kg, sc) enhanced morphine-induced antinociception, with rightward and leftward shift of morphine dose-response curves, respectively. SCH221510 (3mg/kg, sc) combined with morphine (10mg/kg, sc) accelerated the development of morphine antinociceptive tolerance. Conversely, SB612111 (10mg/kg, sc) delayed morphine tolerance development. Neither SCH221510 (3mg/kg, sc) nor SB612111 (10mg/kg, sc) statistically significantly altered the development of morphine-induced physical dependence. Therefore, systemic activation of NOP receptors attenuated morphine antinociception to acute thermal stimuli, facilitated morphine-induced antinociceptive tolerance but did not robustly alter physical dependence in female mice. Systemic blockade of NOP receptors produced opposite actions. These findings demonstrate that N/OFQ-NOP receptor system plays diverse roles in modulating pharmacological profiles of µ-opioid receptor agonists.

NOP receptor antagonism attenuates reinstatement of alcohol-seeking through modulation of the mesolimbic circuitry in male and female alcohol-preferring rats.

In patients suffering from alcohol use disorder (AUD), stress and environmental stimuli associated with alcohol availability are important triggers of relapse. Activation of the nociceptin opioid peptide (NOP) receptor by its endogenous ligand Nociceptin/Orphanin FQ (N/OFQ) attenuates alcohol drinking and relapse in rodents, suggesting that NOP agonists may be efficacious in treating AUD. Intriguingly, recent data demonstrated that also blockade of NOP receptor reduced alcohol drinking in rodents. To explore further the potential of NOP antagonism, we investigated its effects on the reinstatement of alcohol-seeking elicited by administration of the α2 antagonist yohimbine (1.25 mg/kg, i.p.) or by environmental conditioning factors in male and female genetically selected alcohol-preferring Marchigian Sardinian (msP) rats. The selective NOP receptor antagonist LY2817412 (0.0, 3.0, 10.0, and 30.0 mg/kg) was first tested following oral (p.o.) administration. We then investigated the effects of LY2817412 (1.0, 3.0, 6.0 μg/μl/rat) microinjected into three candidate mesolimbic brain regions: the ventral tegmental area (VTA), the central nucleus of the amygdala (CeA), and the nucleus accumbens (NAc). We found that relapse to alcohol seeking was generally stronger in female than in male rats and oral administration of LY2817412 reduced yohimbine- and cue-induced reinstatement in both sexes. Following site-specific microinjections, LY2817412 reduced yohimbine-induced reinstatement of alcohol-seeking when administered into the VTA and the CeA, but not in the NAc. Cue-induced reinstatement was suppressed only when LY2817412 was microinjected into the VTA. Infusions of LY2817412 into the VTA and the CeA did not alter saccharin self-administration. These results demonstrate that NOP receptor blockade prevents the reinstatement of alcohol-seeking through modulation of mesolimbic system circuitry, providing further evidence of the therapeutic potential of NOP receptor antagonism in AUD.

The NOP Receptor System in Neurological and Psychiatric Disorders: Discrepancies, Peculiarities and Clinical Progress in Developing Targeted Therapies

The nociceptin opioid peptide (NOP) receptor and its endogenous ligand nociceptin/orphanin FQ (N/OFQ) are the fourth members of the opioid receptor and opioid peptide families. Although they have considerable sequence homology to the other family members, they are not considered opioid per se because they do not have pharmacological profiles similar to the other family members. The number of NOP receptors in the brain is higher than the other family members, and NOP receptors can be found throughout the brain. Because of the widespread distribution of NOP receptors, N/OFQ and other peptide and small molecule agonists and antagonists have extensive CNS activities. Originally thought to be anti-opioid, NOP receptor agonists block some opioid activities, potentiate others, and modulate other activities not affected by traditional opiates. Because the effect of receptor activation can be dependent upon site of administration, state of the animal, and other variables, the study of NOP receptors has been fraught with contradictions and inconsistencies. In this article, the actions and controversies pertaining to NOP receptor activation and inhibition are discussed with respect to CNS disorders including pain (acute, chronic, and migraine), drug abuse, anxiety and depression. In addition, progress towards clinical use of NOP receptor-directed compounds is discussed.

Differential In Vitro Pharmacological Profiles of Structurally Diverse Nociceptin Receptor Agonists in Activating G Protein and Beta-Arrestin Signaling at the Human Nociceptin Opioid Receptor.

Agonists at the nociceptin opioid peptide receptor (NOP) are under investigation as therapeutics for nonaddicting analgesia, opioid use disorder, Parkinson's disease, and other indications. NOP full and partial agonists have both been of interest, particularly since NOP partial agonists show a reduced propensity for behavioral disruption than NOP full agonists. Here, we investigated the in vitro pharmacological properties of chemically diverse NOP receptor agonists in assays measuring functional activation of the NOP receptor such as guanosine 5'-O-[gamma-thio]triphosphate (GTPγS) binding, cAMP inhibition, G protein-coupled inwardly rectifying potassium (GIRK) channel activation, phosphorylation, β-arrestin recruitment and receptor internalization. When normalized to the efficacy of the natural agonist nociceptin/orphanin FQ (N/OFQ), we found that different functional assays that measure intrinsic activity produce inconsistent levels of agonist efficacy, particularly for ligands that were partial agonists. Agonist efficacy obtained in the GTPγS assay tended to be lower than that in the cAMP and GIRK assays. These structurally diverse NOP agonists also showed differential receptor phosphorylation profiles at the phosphosites we examined and induced varying levels of receptor internalization. Interestingly, although the rank order for β-arrestin recruitment by these NOP agonists was consistent with their ability to induce receptor internalization, their phosphorylation signatures at the time point we investigated were not indicative of the levels of β-arrestin recruitment or internalization induced by these agonists. It is possible that other phosphorylation sites, yet to be identified, drive the recruitment of NOP receptor ensembles and subsequent receptor trafficking by some nonpeptide NOP agonists. These findings potentially help understand NOP agonist pharmacology in the context of ligand-activated receptor trafficking. SIGNIFICANCE STATEMENT: Chemically diverse agonist ligands at the nociceptin opioid receptor G protein-coupled receptor showed differential efficacy for activating downstream events after receptor binding, in a suite of functional assays measuring guanosine 5'-O-[gamma-thio]triphosphate binding, cAMP inhibition, G protein-coupled inwardly rectifying protein channel activation, β-arrestin recruitment, receptor internalization and receptor phosphorylation. These analyses provide a context for understanding nociceptin opioid peptide receptor (NOP) agonist pharmacology driven by ligand-induced differential NOP receptor signaling.

Nociceptin/orphanin FQ neurons in the Arcuate Nucleus and Ventral Tegmental Area Act via Nociceptin Opioid Peptide Receptor Signaling to Inhibit Proopiomelanocortin and A10 Dopamine Neurons and Thereby Modulate Ingestion of Palatable Food

The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits neuronal activity via its cognate nociceptin opioid peptide (NOP) receptor throughout the peripheral and central nervous systems, including those areas involved in the homeostatic and hedonic regulation of energy homeostasis. We thus tested the hypothesis that N/OFQ neurons in the hypothalamic arcuate nucleus (ARC) and ventral tegmental area (VTA) act via NOP receptor signaling to inhibit nearby anorexigenic proopiomelanocortin (POMC) and A10 dopamine neuronal excitability, respectively, and thereby modulate ingestion of palatable food. Electrophysiologic recordings were performed in slices prepared from transgenic male and ovariectomized (OVX) female N/OFQ-cre/enhanced green fluorescent protein-POMC, N/OFQ-cre and tyrosine hydroxylase-cre animals to see if optogenetically-stimulated peptide release from N/OFQ neurons could directly inhibit these neuronal populations. Binge-feeding behavioral experiments were also conducted where animals were exposed to a high-fat-diet (HFD) for one hour each day for five days and monitored for energy intake. Photostimulation of ARC and VTA N/OFQ neurons produces an outward current in POMC and A10 dopamine neurons receiving input from these cells. This is associated with a hyperpolarization and decreased firing. These features are also sex hormone- and diet-dependent; with estradiol-treated slices from OVX females being less sensitive, and obese males being more sensitive, to N/OFQ. Limited access to HFD causes a dramatic escalation in consumption, such that animals eat 25-45% of their daily intake during that one-hour exposure. Moreover, the NOP receptor-mediated regulation of these energy balance circuits are engaged, as N/OFQ injected directly into the VTA or ARC respectively diminishes or potentiates this binge-like increase in a manner heightened by diet-induced obesity or dampened by estradiol in females. Collectively, these findings provide key support for the idea that N/OFQ regulates appetitive behavior in sex-, site- and diet-specific ways, along with important insights into aberrant patterns of feeding behavior pertinent to the pathogenesis of food addiction.

Biased Opioid Ligands.

Achieving effective pain management is one of the major challenges associated with modern day medicine. Opioids, such as morphine, have been the reference treatment for moderate to severe acute pain not excluding chronic pain modalities. Opioids act through the opioid receptors, the family of G-protein coupled receptors (GPCRs) that mediate pain relief through both the central and peripheral nervous systems. Four types of opioid receptors have been described, including the μ-opioid receptor (MOR), κ-opioid receptor (KOR), δ-opioid receptor (DOR), and the nociceptin opioid peptide receptor (NOP receptor). Despite the proven success of opioids in treating pain, there are still some inherent limitations. All clinically approved MOR analgesics are associated with adverse effects, which include tolerance, dependence, addiction, constipation, and respiratory depression. On the other hand, KOR selective analgesics have found limited clinical utility because they cause sedation, anxiety, dysphoria, and hallucinations. DOR agonists have also been investigated but they have a tendency to cause convulsions. Ligands targeting NOP receptor have been reported in the preclinical literature to be useful as spinal analgesics and as entities against substance abuse disorders while mixed MOR/NOP receptor agonists are useful as analgesics. Ultimately, the goal of opioid-related drug development has always been to design and synthesize derivatives that are equally or more potent than morphine but most importantly are devoid of the dangerous residual side effects and abuse potential. One proposed strategy is to take advantage of biased agonism, in which distinct downstream pathways can be activated by different molecules working through the exact same receptor. It has been proposed that ligands not recruiting β-arrestin 2 or showing a preference for activating a specific G-protein mediated signal transduction pathway will function as safer analgesic across all opioid subtypes. This review will focus on the design and the pharmacological outcomes of biased ligands at the opioid receptors, aiming at achieving functional selectivity.

Paranigral VTA Nociceptin Neurons Constrain Motivation for Reward

Nociceptin and the nociceptin opioid peptide (NOP) receptor are widely distributed throughout the brain and have been proposed to have an endogenous role in regulating motivational state; however, how this regulation may occur remains unresolved. NOP receptors are broadly expressed in ventral tegmental area (VTA) dopamine neurons and their activation inhibits dopamine neuron activity, yet the behavioral consequences of this neuropeptide system’s impact in the VTA are unknown. Here we dissected the role of a VTA nociceptin peptide circuit that constrains reward-seeking behavior.

Nociceptin/orphanin FQ modulates energy homeostasis through inhibition of neurotransmission at VMN SF-1/ARC POMC synapses in a sex- and diet-dependent manner

BackgroundOrphanin FQ (aka nociceptin; N/OFQ) binds to its nociceptin opioid peptide (NOP) receptor expressed in proopiomelanocortin (POMC) neurons within the arcuate nucleus (ARC), a critical anorexigenic component of the hypothalamic energy balance circuitry. It inhibits POMC neurons by modifying neuronal excitability both pre- and postsynaptically. We tested the hypothesis that N/OFQ inhibits neurotransmission at synapses involving steroidogenic factor (SF)-1 neurons in the ventromedial nucleus (VMN) and ARC POMC neurons in a sex- and diet-dependent fashion.MethodsElectrophysiological recordings were done in intact male and in cycling and ovariectomized female NR5A1-Cre and eGFP-POMC mice. Energy homeostasis was assessed in wildtype animals following intra-ARC injections of N/OFQ or its saline vehicle.ResultsN/OFQ (1 μM) decreased light-evoked excitatory postsynaptic current (leEPSC) amplitude more so in males than in diestrus or proestrus females, which was further accentuated in high-fat diet (HFD)-fed males. N/OFQ elicited a more robust outward current and increase in conductance in males than in diestrus, proestrus, and estrus females. These pleiotropic actions of N/OFQ were abrogated by the NOP receptor antagonist BAN ORL-24 (10 μM). In ovariectomized female eGFP-POMC mice, 17β-estradiol (E2; 100 nM) attenuated the N/OFQ-induced postsynaptic response. SF-1 neurons from NR5A1-Cre mice also displayed a robust N/OFQ-induced outward current and increase in conductance that was sexually differentiated and suppressed by E2. Finally, intra-ARC injections of N/OFQ increased energy intake and decreased energy expenditure, which was further potentiated by exposure to HFD and diminished by estradiol benzoate (20 μg/kg; s.c.).ConclusionThese findings show that males are more responsive to the pleiotropic actions of N/OFQ at anorexigenic VMN SF-1/ARC POMC synapses, and this responsiveness can be further enhanced under conditions of diet-induced obesity/insulin resistance.

Role of nociceptin/orphanin FQ and nociceptin opioid peptide receptor in depression and antidepressant effects of nociceptin opioid peptide receptor antagonists.

Nociceptin/orphanin FQ (N/OFQ) and its receptor, nociceptin opioid peptide (NOP) receptor, are localized in brain areas implicated in depression including the amygdala, bed nucleus of the stria terminalis, habenula, and monoaminergic nuclei in the brain stem. N/OFQ inhibits neuronal excitability of monoaminergic neurons and monoamine release from their terminals by activation of G protein-coupled inwardly rectifying K+ channels and inhibition of voltage sensitive calcium channels, respectively. Therefore, NOP receptor antagonists have been proposed as a potential antidepressant. Indeed, mounting evidence shows that NOP receptor antagonists have antidepressant-like effects in various preclinical animal models of depression, and recent clinical studies again confirmed the idea that blockade of NOP receptor signaling could provide a novel strategy for the treatment of depression. In this review, we describe the pharmacological effects of N/OFQ in relation to depression and explore the possible mechanism of NOP receptor antagonists as potential antidepressants.

ERK and p38 contribute to the regulation of nociceptin and the nociceptin receptor in human peripheral blood leukocytes.

Little is known about the mechanisms involved in the regulation of nociceptin and its receptor (nociceptin opioid peptide receptor, NOP) in response to inflammation and pain in humans. In this study, specific signaling pathways contributing to the regulation of nociceptin and NOP in human peripheral blood leukocytes were investigated. After approval by the ethics committee, peripheral blood obtained from healthy donors was cultured with or without phorbol-12-myristate-13-acetate (PMA). Prepronociceptin (ppNOC) and NOP mRNA were analyzed by real-time quantitative polymerase chain reaction, and nociceptin concentrations in culture supernatants by fluorescent enzyme immunoassay. Nociceptin and NOP protein levels in blood leukocyte subsets were determined using flow cytometry. To examine the contribution of signaling pathways to ppNOC and NOP regulation, blood was pre-treated with kinase inhibitors specific for ERK, JNK, p38, and NFκB pathways prior to culturing with or without PMA. PMA dose-dependently upregulated ppNOC mRNA but downregulated NOP mRNA in human peripheral blood leukocytes. PMA 10 ng/ml increased ppNOC after 6 h and suppressed NOP after 3 h compared to controls (both P <0.005). Nociceptin concentrations were increased in supernatants of PMA-induced blood samples after 24 h (P <0.005), whereas expression of cell-membrane NOP was decreased by PMA in blood leukocyte subsets (all P <0.05). Blockade of ERK or p38 pathways partially prevented PMA effects on ppNOC and NOP mRNA (all P <0.05). The combination of ERK and p38 inhibitors completely reversed the effects of PMA (P <0.05). ERK and p38 are two major signaling pathways regulating nociceptin and its receptor in human peripheral blood leukocytes under inflammatory conditions.

Pain Wars: A New Hope

Nociceptin opioid peptide receptor agonists interact with mu-opioid receptor agonists for pain relief. A new study by Ding etal. (2018) examines a bifunctional nociceptin- and mu-opioid receptor agonist, AT-121, that provides analgesia without physiological side effects or abuse liability, offering a promising new hope toward better analgesics.

Signaling Pathways Contributing to Central Kappa Opioid Diuresis

Central activation of alpha‐2 receptors and nociceptin opioid peptide receptors (NOP) produce marked diuresis in rats, which is mediated in part by a downstream G‐alpha z protein signaling pathway that inhibits antidiuretic hormone (ADH) secretion. These studies investigated whether central G‐alpha protein signaling pathways also contribute in producing the diuretic and/or antinatriuretic response to central administration of the kappa opioid agonist, U‐50488H. Sprague Dawley rats were implanted with an intracerebroventricular (i.c.v.) cannula and pretreated with vehicle or pertussis toxin (PTX), which inhibits the Gi/o family of proteins except for Gz. After 48‐hrs pretreatment, rats were instrumented with cannula in the femoral artery, vein, and bladder and infused i.v. with isotonic saline (55 μl/min). After recovery, blood pressure (BP), heart rate (HR), and urine output (V) were collected in conscious rats before (control) and for 90‐min after (10‐min collections) i.c.v. U‐50488H (n=6). In vehicle pretreated rats, i.c.v. U‐50488H produced a marked increase in V (max Δ, 101±8 μl/min) and decrease in urinary sodium excretion (UNaV; max Δ, 5±0.5 μeq/min). The diuretic response produced by U‐50488H was significantly (p&lt;0.05) blunted by pretreatment with PTX (max Δ, 57±3 ul/min). In contrast, PTX did not alter the decrease in UNaV. U‐50488H did not alter BP or HR in either group. To explore which G‐alpha proteins may participate in the kappa opioid mediated diuresis, separate rats were pretreated i.c.v. (24‐hrs) with an oligodeoxynucleotide (ODN), which selectively targeted a specific G‐alpha protein. In these studies, pretreatment of groups of rats (n=6/group) with a single ODN for either Gi1, Gi2, Gi3, Go, or Gz, failed to blunt the maximal diuresis as compared to the increase in V produced by i.c.v. U‐50488H in scrambled ODN treated rats. These results demonstrate that central kappa opioids produce a pertussis toxin sensitive diuresis, but not antinatriuresis. This suggests that a combination of Gi/o proteins may be required to produce the aquaretic effects of kappa opioid agonists.Support or Funding InformationSupported by NIH P30GM106392 to DRKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Occupancy of Nociceptin/Orphanin FQ Peptide Receptors by the Antagonist LY2940094 in Rats and Healthy Human Subjects.

Therapeutic benefits from nociceptin opioid peptide receptor (NOP) antagonism were proposed for obesity, eating disorders, and depression. LY2940094 ([2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4'-piperidine]-1'-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol) is a novel, orally bioavailable, potent, and selective NOP antagonist. We studied NOP receptor occupancy (RO) after single oral LY2940094 doses in rat hypothalamus and human brain by use of liquid chromatography with tandem mass spectrometry (LC-MS/MS) (LSN2810397) and positron emission tomography (PET) ([(11)C]NOP-1A) tracers, respectively. A bolus plus constant infusion tracer protocol with PET was employed in humans at 2.5 and 26.5 hours after administration of the LY2940094 dose. The RO was calculated from the change in regional distributional volume (VT) corrected for nondisplaceable volume using Lasson plots. The RO followed a simple Emax relationship to plasma LY2940094 concentration, reaching near complete occupancy in both species. For rat hypothalamus, the plasma concentration at half-maximum RO (EC50) was 5.8 ng/ml. In humans, LY2940094 was well tolerated and safe over the 4-40 mg dose range, and it peaked in plasma at 2 to 6 hours after a 1- to 2-hour lag, with approximate dose-proportional exposure. After 4-40 mg doses, NOP RO was similar across the prefrontal cortex, occipital cortex, putamen, and thalamus, with EC50 of 2.94 to 3.46 ng/ml, less than 2-fold lower than in rats. Over 4-40 mg doses, LY2940094 mean plasma levels at peak and 24 hours were 7.93-102 and 1.17-14.1 ng/ml, corresponding to the cross-region average NOP RO of 73%-97% and 28%-82%, respectively. The rat EC50 translates well to humans. LY2940094 readily penetrates the human brain, and a once-daily oral dose of 40 mg achieves sustainably high (>80%) NOP RO levels suitable for testing clinical efficacy.

Design, synthesis and evaluation of [3H]PF-7191, a highly specific nociceptin opioid peptide (NOP) receptor radiotracer for in vivo receptor occupancy (RO) studies

Herein we report the identification of (+)-N-(2-((1H-pyrazol-1-yl)methyl)-3-((1R,3r,5S)-6′-fluoro-8-azaspiro[bicyclo[3.2.1]octane-3,1′-isochroman]-8-yl)propyl)-N-[3H]-methylacetamide {[3H]PF-7191 [(+)-11]} as a promising radiotracer for the nociceptin opioid peptide (NOP) receptor. (+)-11 demonstrated high NOP binding affinity (Ki=0.1nM), excellent selectivity over other opioid receptors (>1000×) and good brain permeability in rats (Cb,u/Cp,u=0.29). Subsequent characterization of [3H](+)-11 showed a high level of specific binding and a brain bio-distribution pattern consistent with known NOP receptor expression. Furthermore, the in vivo brain binding of [3H](+)-11 in rats was inhibited by a selective NOP receptor antagonist in a dose–responsive manner. This overall favorable profile indicated that [3H](+)-11 is a robust radiotracer for pre-clinical in vivo receptor occupancy (RO) measurements and a possible substrate for carbon-11 labeling for positron emission tomography (PET) imaging in higher species.